WO1993012776A1 - Aids virus infection inhibitor composition - Google Patents

Abstract

D-β-Lysylmethanediamine represented by formula (I) or a salt or hydrate thereof has an activity of inhibiting the infection with AIDS virus or HIV, and hence an AIDS virus infection inhibitor containing the same as an active ingredient is prospective as one of the remedies for AIDS which is thought to be difficult to cure.

Description

 明 Description

 AIDS infection inhibiting composition

 Technical field

 The present invention relates to a human acquired immune deficiency syndrome virus comprising D-lysyl methazine diamin or a salt or hydrate thereof as an active ingredient. In other words, the present invention relates to a composition for inhibiting the infection of AIDS virus (hereinafter sometimes abbreviated as IV). Further, the present invention provides an AIDS virus comprising treating human cells with D-base-lysyl-mesyldiamine, a salt or hydrate thereof. Human — about methods of inhibiting cell infection. Further, the present invention relates to the use of D-base-lysylmethazinediamine, a salt or hydrate thereof, for preparing an antiviral composition for inhibiting AIDS virus infection. And about.

Background art

 AIDS is a disease caused by infection of human cells with HIV, which has become a social problem. Several drugs that inactivate HIV have been reported, but none have been satisfactory as AIDS treatments. Therefore, there is a current need to provide new, less toxic antiviral agents that have potent activity in inhibiting human cells from becoming infected with HIV. You.

On the other hand, D-beta-lysyldiamine obtained previously by the present inventors is an antibacterial substance having weak antibacterial activity against Gram-positive bacteria. D—Overnight-Resin temperature is calculated by the following formula (I)

 (R)

H ₂ CH ₂ CH ₂ CH ₂ CHCH ₂ C0NHCH2NH ₂ (I)

 I

NH ₂

Microorganisms, Streptomyces ■ Produced by Streptomyces nashvii 11 ensis and have been [Journal of Obv Anticipix J 39, 3, p. 476 (1986), and Tokukai Sho 62] — 114947, published on May 26, 1987.) D—Beta-Risylmethadiamamine 1/2 carbonate is a hygroscopic, colorless powder with a distinct melting point. Its specific rotation is [α] _D ^{2 S} = —7.4 ° (c 0.5, water).

 An object of the present invention is to provide a novel anti-AIDS drug that strongly inhibits the infection of human T cells by HIV and has low toxicity to mammals. To provide effective anti-HIV agents. Another object of the present invention is to provide a novel pharmaceutical composition that inhibits AIDS virus infection. Yet another object of the present invention is to provide a pharmaceutical method for inhibiting human T-cell infection by HIV by treating the human T-cell with an antiviral agent. It is in. Other objects of the present invention will become clear from the description below.o

Disclosure of the invention

In order to achieve the above object, the present inventors have conducted various studies. As a result, the present inventors have found that D—beautiful—lysyl methadiamamine, a salt or hydrate of which is a human T due to HIV. For the first time, it was discovered that it has high activity in blocking single-cell infection, but has low toxicity to mammals. That is, according to the experiments of the present inventors, it was found that D-beta-glycidylmethandiamin, a salt or hydrate thereof, was previously treated in a medium. When the cultured human T cells are brought into contact with HIV by adding HIV thereto, the infection of the human T cells by HIV becomes D—between—lysyl. It is recognized that inhibition can be achieved by the action of Ndamine, its salts or hydrates. Therefore, D-base-lysylmethazinediamine, its salt or hydrate, has potent and broad activity in inhibiting HIV infection of human T-cells. In a sense, it has antiviral activity against HIV.

 Therefore, in the first gist of the present invention, the following formula (I)

(R)

H ₂ NCH ₂ CH ₂ C H2C HCH ₂ C ONHCH ₂ NH ₂ (I)

Formulated with a pharmaceutically acceptable carrier that contains D-baseyl lysylmethanediamin or its salt or hydrate represented by NH ₂ as an active ingredient And a composition for inhibiting HIV infection.

In still another aspect, the present invention provides a method for inhibiting the infection of human T-cells by AIDS virus, comprising the step of treating a D-baseyl radical of formula (I) with an effective amount. Amin, its pharmaceutically acceptable A method of inhibiting the infection of human T-cells by AIDS virus, comprising treating the human T-cells with acceptable salts or hydrates.

 In another aspect, the present invention relates to a method for producing a pharmaceutical composition for inhibiting AIDS virus, which comprises preparing a D-beta-ridylmethazinediamine of the formula (I): And the use of commercially acceptable salts or hydrates.

 Furthermore, the present invention relates to a compound of the formula (I) comprising a pharmaceutically acceptable salt or hydrate of D-beta-glycidylmethazinediamine of the formula (I). Also, the present invention relates to a method for producing a pharmaceutical composition for inhibiting AIDS virus infection, which comprises mixing with a liquid carrier.

 BEST MODE FOR CARRYING OUT THE INVENTION

 Since D-lysylmethanediamine used as an active ingredient or an active compound in the present invention is a water-soluble basic substance, an organic or inorganic acid and an acid addition salt are used. Can be formed. Pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and nitric acid, as well as acetic acid, lingoic acid, cuenic acid, There are pharmaceutically acceptable organic acids such as asconolevic acid and methansulfonic acid.

The D-betadilylmethazine diamine used in the present invention is characterized by having low toxicity to mammals. For example, in an acute toxicity test by intravenous administration in mice, mice were able to withstand a dose of 250 mg kg of D-beta-lysylmethandiamine. There were no deaths.

 It contains D-beta-Lidinolemethandiamine or its acid addition salt or hydrate as its active ingredient, which is a pharmaceutically acceptable solid or The pharmaceutical composition of the present invention formulated with a liquid carrier can be prepared into various forms by a conventional method. The AIDS infection inhibitor composition of the present invention thus prepared can be formulated into a conventional dosage form such as a powder, granule, tablet or syrup, or injection. It can be administered orally or parenterally.

 Next, we demonstrate that D—Ridylmethazine diamine has activity to inhibit the transmission of human T—cells by HIV, or AIDS virus. In order to do this, the following test examples were performed. The test method is as follows.

Test example 1

 The effect of HIV T on the infection of human T-cells by D-beta-glycinol methanediamine is described in "Pro Natl. Acad Sci. USA," 80, 6061-6065 ( 1983); "J. Antibiot.", 40, 1077-1078, (1987); "J. Antibiot.", 42, 344-346, (1989) and "J. Antibiotno", 44, 1228-1236, (1991) according to the evaluation method.

That is, each of the co scan te flop les over bets 48 holes, Ru kind der of T cells-suspension have been human in phosphate buffer Micromax T - 4 cells 1 X 10 ⁵ cells / ^/ a And 0.5? ^ Per hole. As a test compound, D-beta-methylmethamine diamine is dissolved in a phosphate buffer solution for 3, 10, 30 and 100 0.05 ^ of a solution containing μ. gZ ^ was added to each well. After incubating the cells for 2 hours under 5% CO2 at 37 ° C, HIV (HTLV-Ea strain) was added to each well.

(m.0.i) corresponding to the amount of virus of 0.025-0.05] to infect MT-4 cells. Further, the plate was kept at 37 ° C under 5% CO 2 for 4 days, and the above T-cells were treated in the presence of each concentration of the test compound.

A part of the culture was taken, MT-4 cells were spread on slide glass, the cells were dried and fixed with acetate. The expression (presence) of HIV-4 cells positive for HIV antigen was determined by indirect immunofluorescence (Y. Hinuma et al., "Proc. Natl. Acad. Sci. USA," 78, 6476-6480 (1981) and Υ · Takeuchi Et al., "Gann," 78, 11-15 (1987)). For this, the above-mentioned smear and the cell smear fixed with acetate are used as a primary antibody using the serum of an AIDS patient diluted 10-fold with a phosphate buffer. Treated at ° C for 30 minutes. After the treatment, the MT-4 cells were washed with a phosphate buffer, and then conjugated with fluorescein • isothiosinate and egret anti-human IgG serum (Cappel Laboratories). , Cochranrile, PA, USA) as a secondary antibody at 37 ° C for 30 minutes. Further, the cell smear was washed with a phosphate buffer and covered with a cover glass, and the cells were examined under a fluorescence microscope. The percentage of the number of virus antigen-positive cells (ie, cells expressing an HIV-specific antigen and emitting fluorescence) per the total number of cells was calculated. In addition, the above test was repeated in the same manner without the addition of D-lysylmethandiamine, and was used as a control test.

 We also measured the cytotoxicity of D-lysylmethandiamine on MT-4 cells. — Incubate MT-4 cells under the same culture method and conditions as in the HIV infection inhibition test described above, in which risinolemethandiamin is present at various concentrations but no HIV is added. I went by doing. If no cytotoxicity is observed, it is indicated by the symbol-in Table 1 below.

 In order to evaluate the inhibitory activity of human T-cells, that is, MT-4 cells, by HIV, on D-vessell-lysylmethandiamin, the test compound was used to evaluate its inhibitory activity. The culture of MT-4 cells in the presence of the test compound relative to the number of H-factor V antigen-positive cells (C) measured in the control test described above, in which the culture of MT-4 cells was performed by addition, was performed. The percentage (TZC,%) of the number (T) of HIV antigen-positive cells measured in the HIV infection inhibition test described above was calculated. The results of the calculation are shown in Table 1 below as the expression rate of HIV antigen-positive cells ().

1

 Test compound concentration

 3 10 30 100

 (i g / m 1)

 HIV antigen positive cells

30 15 10 5 Expression rate (%) Cell toxic As is clear from the test results in Table 1 above, the D-beta-lysylmethadiamamine used in the present invention has no cytotoxicity even at a concentration of 100 / g, It was confirmed that the concentration of HIV virus antigen-positive cells was significantly reduced at a concentration of / gZrng or less, that is, the expression of HIV virus antigen was significantly suppressed. 50% effective concentration (ED ₅ Q) is 0.62 H g / id der one Na:.

 This is due to the fact that HIV cannot proliferate in human MT-4 cells due to the presence of D-beta-dilylmethanediamin, and that HIV virus antigens cannot be expressed in MT-14 cells. This indicates that expression was suppressed.

 According to another recent experiment, at a concentration of 100 g Z, D-beta-dilylmethazine diamin neither inhibits HIV reverse transcriptase nor inhibits HIV protease. It did not show any activity. Therefore, it was found that the infection of D-beta-lysylmethazinediamine HIV was more strongly inhibited by an unclear mechanism.

By the way, recently AIDS therapeutic agents to DDI that Tsu Do Ni Let 's that are used (2', 3 'over Jidokishii raised to the power of emissions) and intends at the same time line the same test that had use of, DDI of ED _5. The value was found to be 0.46 £ g Z, and DDI shows the same effect of suppressing T-cell infection by HIV as D-beta-lysylmethanediamin of the present invention. I did.

Practically administered D-betadilylmethazinediamine as an HIV infection inhibitor or inactivator according to the present invention. To do so, they can generally be used orally or parenterally.

 The active compound in the present invention, that is, D-base-lysyl methazine diamine, its salt or hydrate alone or in combination with an excipient or carrier They are mixed and administered in the form of injections, oral preparations, or suppositories. Excipients and carriers are selected from those that are pharmaceutically acceptable, and their type and composition are determined by the route and method of administration. For example, water, alcohol or animal or vegetable oils such as soybean oil, sesame oil, mineral oil, or synthetic oil, or synthetic oils may be used as the liquid carrier. As solid carriers, sugars such as maltose and sucrose, amino acids such as lysine, cellulose derivatives such as hydroxypropyl propyl cellulose and cycle derivatives Polysaccharides such as rodextrin and organic acid salts such as magnesium stearate are used.

When formulated as an injection, physiological saline, various buffers, saccharide solutions such as glucose, inositol and mannitol, and ethylene glycol are generally used. And glycols such as polyethylene glycol are desirable. In addition, sugars such as inositol, mannitol, glucose, mannose, maltose, sucrose, and phenylalanine A lyophilized preparation together with excipients such as amino acids and a suitable solvent for injection at the time of administration, such as sterile water, saline, glucose solution, electrolyte solution, and amino acids. It can be used by dissolving it in any liquid for intravenous administration. The content of D-beta-lysylmethazinediamine in the formulated composition varies depending on the formulation type, but is usually 0.1 to 100% by weight, preferably 1 to 100% by weight. 90% by weight. For example, in the case of an injection solution, it is usually preferable to contain 0.1 to 5% by weight of the present compound. In the case of oral administration, it is used in the form of tablets, capsules, powders, granules, dry syrups, liquids, syrups, etc. together with the above-mentioned solid or liquid carriers. In the case of capsules, tablets, granules and powders, the content of the compound is generally 3 to 100% by weight, preferably 5 to 90% by weight, and the balance is a carrier.

 D—The dose of beta-methylmethamine is determined by the patient's age, weight, symptoms, and the purpose of treatment. Therapeutic dose is generally non-permanent. 1 to 100 mg kg / day, and oral administration 5 to 500 mgr Z kg / day. However, the dose can be administered continuously or intermittently within a range that does not exceed a certain amount in consideration of various situations such as the results of animal tests.

 In the case of parenteral administration, the total dose may be appropriately changed according to the administration method, patient condition, for example, age, body weight, sex, diet, concomitant medication, etc. . Appropriate dosages and frequency of administration under certain conditions should be decided by a specialist based on the above measures. These administration conditions are the same for oral administration.

Next, preparation examples of the HIV infection-inhibiting composition of the present invention are shown. Formulation Example I D — Beta — 10 g of lysylmethazine diamine hydrochloride is added to purified water and dissolved to form a solution of 100 ^. Then, use a Millipore filter GS type. It was sterilized and filtered. Each of the filtrates is poured into 5 vials, lyophilized, and freeze-dried injectable containing 1 mg of D-beta-lysylmethane diamine hydrochloride lOOmg per vial. I got

Formulation Example 2

 D — Beta — Mix well 5 g of lysinolemethandiamamine hydrochloride, 60 g of lactose, 33 g of crystalline cellulose and 2 g of hydroxypropyl propylcellulose. Then, it was compressed using a roll-type compressor (roller compactor) to crush the compressed material. The crushed material was sieved to collect the fractions between 16 mesh and 60 mesh and collected as granules.

Formulation Example 3

 3 g of D _ beta-glycinol dimethylamine, 12 g of crystalline lactose, 15 g of crystalline cellulose and 0.3 g of magnesium stearate The mixture was tableted with a mixing machine to obtain one tablet containing 500 mg of D-base-lysylmethanediamin.

D — The production of beta-methylmethane dimamine is carried out by using the microorganism Streptomyces nassieuvirensis MD743-GF4 by ordinary microorganisms. By inoculating such a nutrient-containing medium, culturing, and growing under favorable or aerobic conditions, the D-beta-diphenylmethane is mainly contained in the culture medium. By producing and accumulating amines, and further by separating the target substance from the culture, especially the culture broth or culture filtrate. It is.

 As a nutrient source in the medium used for the culture, a known carbon source or nitrogen source used as a nutrient source for actinomycetes can be used. , Peanut flour, cottonseed flour, dried yeast, peptone, meat extract, potato zein, corn 'Steep' liquid, sodium nitrate, ammonium sulfate, etc. Carbon sources that can be assimilated include commercially available carbohydrates such as glucose, galactol, starch, glycerin, maltose, dextrin, sucrose, and lactose. Or soybean oil, fat, etc. If necessary, inorganic salts such as salt, calcium carbonate, magnesium sulfate, manganese chloride, ammonium sulfate, phosphates, and various amino acids can be used. D-All night-D-beta-Rigyl tandiamine-producing bacteria can be used, and D-beta-Rigyl tandiamine can be used for production. All materials can be used for cultivation of the above-mentioned MD744-GF4 strain.

In culturing the above-mentioned strain MD7431-GF4, a submerged liquid aeration and stirring culture is preferable, and the culture temperature is D. The culture temperature is D. , D—overnight—Risylmethane Diamine can be selected within a range of concentrations, with 25-30 ° C being particularly preferred. Culture of the strain MD 7 4 3-GF 4 is usually continued until D-sufficient amount of lysyldiamine is accumulated in the culture. Usually, cultivation of bean is carried out for 2 to 7 days. D _ beta-lysinolemethandiamin and its acid addition salts or hydrates are soluble in water, and cultivate D-beta-lysylmethazine diamin-producing bacteria. Mainly present in the liquid part of an object. This is because D-beta-lysylmethazinediamine in the liquid portion of the culture is virtually insoluble in organic solvents such as butanol, butyl acetate, and cross-linked form. Treatment with these organic solvents can be used if necessary to remove unwanted compounds or contaminants from the culture filtrate. Activated carbon is used as an adsorbent that can be collected by adsorption using various adsorbents for D-base in a culture solution or an aqueous solution. In this case, the adsorbed D-beta-diylmethanediamin is eluted with weakly acidic water and weakly acidic organic, propanol, and acetate water. By this, it is desorbed from the activated carbon.

In addition, because of its basicity, D-base-lysylmethazinediamine is adsorbed on the cation-exchange resin in good yield and eluted with a suitable eluent. The cation exchangers used for this purpose include amberlite IRC-150, which has a carboxyl group as an active group, and amino-lite CG-150 (Rome-Anne). de Nono - made, Inc.), Les word Ji Tsu door CNP (manufactured by Bayer Co., Ltd.), CM - Se off a de-click scan (made off Alma shea Co., Ltd.) of any H-type, N a type, NH ₄ There is a positive ion exchange resin such as a mold, or a mixture thereof. Antibiotics adsorbed on the cation exchanger are eluted with acidic water, dilute ammonia water and aqueous solutions of inorganic salts in good yields, usually 0.2 N-1 N hydrochloric acid and 0.5 N—2 N ammonia water is used. Since D-beta-methyldiamine diamin does not substantially adsorb to the anion-exchange resin, this means neutralization of the acidic solution containing the compound or acidification from the compound. Can be used to remove contaminants.

 D-Before-Lilymethane Diamine is obtained by appropriately combining the extraction and separation methods described above, or by using one or more of these methods. The microbiological properties of Streptomyces nasshuvirensis MD743-GF4 strain, which is a bacterium producing diamin, are described in Japanese Patent Application Laid-Open No. 62-114947. It is described in detail in the description. The following is a summary of the mycological properties of this strain MD74—GF4.

 The MD7443-1 GF4 strain forms a spiral aerial mycelium from the basal hypha separated under a microscope, and a rotative technique cannot be observed. The mature spore chain has a chain of 20 or more spores, the size of the spore is about 0.6 to 0.7 X I.4 to 1.7 micron, and the spore surface is smooth. On various media, the strain MD743-3-0-4 develops colorless to light yellow tea, or white to light brown ash aerial hyphae on the development of light brown tea and soluble color. The element is barely noticeable or slightly yellowish. The melanin-like dye is positive, the proteolytic power is moderate, and the starch has a stronger water-decomposability.

The MD7 4 3 — GF4 strain was transformed by Streptococcus-Nasch Virgensis IMCS-0342 (ISP 5314) (international Journal of Systematic Bacteriology, 19, p. 455, 1969), the MD743-GF4 strain does not show any use of D-xylose, but the IMCS-0342 strain (ISP5314) has D-xylose. This is in good agreement with the latter except that it indicates the use of a source.

 The above-mentioned Streptomyces nutsius biluensis MD743-GF4 strain was deposited with the Ministry of International Trade and Industry of Japan, the Ministry of International Trade and Industry, and the Research Institute of Microorganisms and Industrial Technology on September 7, 1985. (FERMP — 8442), and was re-deposited on December 25, 1991, and subsequently deposited under accession number FERMP — 12676J. Currently MD 74 3 — GF 4 strain The deposit was transferred under the terms of the Vedapest Treaty and deposited with the Institute of Fine Arts under the deposit number "FERMBP-4127". We are located in Tsukuba City, Ibaraki Prefecture.

 Reference Example 1 shows an example of an enzymatic production of D-beta-glycidylmethamine.

Reference example 1

Streptomyces nasshu virgensis MD 743 — GF4 strain cultured on an agar slant culture medium (Microbe Kencho, No. 12676, Microbe Kenjo, No. 4127) , Galactose 2.0%, dextrin 2.0%, peptone (Bacto-Sonton, made by Difco) 1.0%, cone 'step-up' recovery (Made of Ajinomoto) Liquid medium containing 0.5%, ammonium sulfate 0.2%, and calcium carbonate (inoculated in 500% triangular flask, rotated at 28 ° C for 2 days) A seed culture was obtained by shaking culture (rotation at 180 rpm). A medium (110) having the same composition was inoculated, and 90 triangular flasks were cultured under the same conditions for 3 days.

を得た。 こ の濾液を陽イ オ ン交換樹脂、 ア ンバーラ イ ト I R C — 50 ( N H 4 型 ： H型の混合物、 7 ： 3 ) 550 を充填 した塔に通過、 D — べ一タ ー リ ジ ルメ タ ン ジァ ミ ンを吸着せ しめた。 樹脂塔を水洗（1， 100 i ) 後、 1.2 Nア ン モニア水で溶出 した。 溶離液の活性 面分 を集めて減圧下に濃縮乾燥 し、 粗粉末 636 mg ( 644 g / mg) を得た。 This culture was collected, filtered and filtered to 9000 filtrates (pH 6.0, titer 80 zg).

I got This filtrate is passed through a column packed with cation exchange resin, Amberlite IRC-50 (NH4 type: H-type mixture, 7: 3) 550, D-beta-Rigid meter The diamine was adsorbed. After the resin tower was washed with water (1,100 i), it was eluted with 1.2 N ammonia water. The active surface of the eluate was collected and concentrated and dried under reduced pressure to obtain 636 mg (644 g / mg) of a crude powder.

の水に とか し、 その水溶液を陽ィ ォ ン交換樹脂ア ンバ一ラ イ ト 60m£の塔に通 して活性化合物 を吸着さ せた。 次に、 こ の樹脂塔を水 120? ^で洗浄 し (その流出液は分画 1 — 18と して集める） 、 さ らに 6 N ア ンモニア水 300 で洗浄 した （その流出液は分面 19一 60と して集める） 。 最后に、 塔を 1.2Nア ンモニア水 300 で溶出 して溶出液を分面 61— 101と して収得 した。 This coarse powder

The water was dissolved in the solution, and the aqueous solution was passed through a column of 60 ml of anion-exchange resin amberlite to adsorb the active compound. Next, the resin tower was washed with 120 120 of water (the effluent was collected as fractions 1 to 18) and further washed with 300 of 6N ammonia water (the effluent was separated). Collected as 19-1 60). Finally, the column was eluted with 1.2N ammonia water (300), and the eluate was obtained as a separation plane 61-101.

Combine the separation surfaces 77-95, concentrate to dryness under reduced pressure, and dry further to obtain D-base resin, a half-carbonate of glycinolethanediamine, a colorless powder. Obtained as a substance (hygroscopic). The yield was 370 mg (titer: 1,000 g /), and the yield from the culture filtrate was 50-8%. This substance is a specific rotation of [] ^{D 2 6 - 7.4 ° (c} 0.5, water) was shown. This substance became colorless when absorbed. Industrial applicability

 As described above, according to the present invention, D-base-lysylmethazinediamine, its salt or hydrate is used for AIDS (human acquired immunodeficiency syndrome) virus, It was found to have HIV infection inhibitory activity. An AIDS infection inhibitor containing this compound or its salt or hydrate as an active ingredient is provided, and it is one of the remedies for AIDS, which is currently considered difficult to treat. Is expected.

Claims

The scope of the claims  1. The following equation (I) "(R) H ₂ NCH ₂ CH ₂ CH ₂ CHCH ₂ CONHCH ₂ NH ₂ (I) D represented by NH _2- containing diazine or its salt or hydrate as an active ingredient and containing a pharmaceutically acceptable carrier A virus, a composition that inhibits the transmission of HIV.  2. An effective amount for inhibiting the infection of human T-cells by AIDS virus, D-formyl lysylmethanediamine of formula (I) according to claim 1 or a preparation thereof. A method of inhibiting human T-cell infection by AIDS virus, comprising treating human T-cells with a biologically acceptable salt or hydrate.  3. In the manufacture of a pharmaceutical composition for inhibiting AIDS virus infection, D-butyrylmethazinediamine of formula (I) according to claim 1 and its pharmaceutical preparation Use of acceptable salts or hydrates.  4. The pharmaceutically acceptable salt or hydrate of D-betasyldimethazinediamine of formula (I) according to claim 1 is pharmaceutically acceptable. A method for producing a pharmaceutical composition for inhibiting HIV infection, comprising mixing with a solid or liquid carrier.